Bacteria able to produce and secrete proteins encoded by heterologous genes are used extensively for the industrial production of high value-added pharmaceutical proteins such as human and animal growth hormones, insulin, interferons, cytokines etc. Organisms other than E. coli thus far used or proposed for industrial production include cultured mammalian and insect cells, yeasts and fungi, and a number of Bacillus spp. Among the bacteria already widely used for industrial purposes are the lactic acid bacteria, which are employed as starter cultures for fermented food-stuffs, and as flavour enhancers, and preservatives. These properties depend on the ability of these organisms to produce certain enzymes, lactic acid and harmless antimicrobial polypeptides such as nisin. To date only low yields of foreign proteins have been obtained by the genetic manipulation of these organisms, and in some instances gene expression has depended on the use of unregulated genes, or of undefined control elements. Lactic acid bacteria which are related to those used in food and milk fermentations are also found as commensal bacteria in the alimentary tracts of animals. There is considerable industrial interest in the genetic manipulation of both the food and the commensal bacteria. For example, recombinant strains of these bacteria could be used to improve fermentation processes, and as novel vectors for multi-disease vaccines.
In contrast to a Gram-negative organism such as E. coli Gram-positive bacteria such as the lactic acid bacteria and Bacillus spp. have the capacity to secrete proteins more readily into the growth medium. However, the active protease systems of the best known bacillus species, B. subtilis, have greatly limited the usefulness of this organism for the production of recombinant proteins. Protein secretion in Gram-positive cells differs fundamentally from that observed in Gram-negative cells, where it is a complex two stage process in which true secretion (as opposed to protein accumulation in the periplasmic space) requires that exported proteins should traverse both the cell membrane and the outer membrane. Thus, although large amounts of recombinant proteins can be produced in E. coli many of these proteins become insoluble and inactive, and either accumulate within the cytoplasm, or are secreted as far as the periplasm, where they may precipitate, and lose their biological activities. For many such recombinant proteins renaturation procedures for the recovery of biological activity are an expensive and difficult aspect of downstream processing. For these reasons the use of naturally secretory organisms for protein production may be highly advantageous.
As a separate aspect of the development of bacterial technologies, recombinant vaccine strains of certain pathogenic bacteria (mycobacteria, salmonella) have been proposed for the production and delivery in vivo of protective protein antigens of a range of disease-causing bacteria, viruses, and protozoan and metazoan parasites. However, even attenuated vaccine strains of pathogenic bacteria are to some degree invasive, and the immune responses generated by these organisms may result in immunopathological damage. Development of non-invasive microbes in forms suitable for effective antigen-presentation to the immune system would provide previously unattainable levels of vaccine safety. In particular, if given by the oral route, such organisms might stimulate the mucosal immune system preferentially, thus providing a basis for the development of live, oral vaccines which would protect against infection. Alternatively or additionally these vaccines might be given by injection, or administered orally or by injection to boost immune responses primed with recombinant mycobacteria or salmonella; in this instance the innate differences between the antigenic constituents of the priming vaccine carrier and those of the booster could be expected to minimise immunopathological damage, and to boost immune responses to the expressed recombinant antigens preferentially. Furthermore, the capacity to express a range of foreign proteins in non-invasive microorganisms opens the way to the concurrent delivery of antigens and cytokines, which might be used to drive an immune response in a desired direction. The successful development of all these applications requires that a regulated system for high level foreign gene expression should be available for use in lactic acid bacteria.
Although there are several reports of the expression of foreign genes in L. lactis none of these describes either a regulated system, or the production of substantial quantities of protein. In two of these cases antibiotic resistance genes were used as reporter genes to identify secretion signal sequences and/or promoter sequences.sup.1,2. The other five cases comprise two proteins of eukaryotic origin and three prokaryotic proteins of Gram-positive origin. The eukaryotic proteins concerned are hen egg white lysozyme and bovine prochymosin. The prokaryotic proteins are Bacillus subtilis neutral protease, the .beta.-galactosidase of Clostridium acetobutyicum and the pAC protein antigen of Streptococcus mutans. The results obtained with these latter three proteins provide the best comparison with our own work, since we have also used as a model system one which involves the expression of a protein of Gram-positive origin (the tetanus toxin fragment C). However, our own results are unique, in that we have devised a system of regulated gene expression and product secretion, and also obtained significant yields of the expressed protein product, whether secreted or not. The previous studies have resulted in only low and (with one exception) undetermined amounts of foreign protein being formed, whereas the system we have developed has reliably yielded 3.4% of soluble cytoplasmic protein as the desired product with a secretory expression system, and 22% of soluble cytoplasmic protein with a non-secretory expression system. In addition, with the secretory system, product is secreted into the supernatant in a progressive fashion, reaching an estimated final yield (under test-tube conditions) of 5-10 mg/L.
In the studies cited above hen egg white lysozyme.sup.3 was expressed as a fusion protein which either lacked activity or was produced in too low an amount to be detected in the assay used. Biologically active Bacillus subtilis neutral protease.sup.4 was expressed and secreted from L. lactis using either its own promoter or a lactococcal promoter. The amounts of neutral protease produced (in arbitrary units) were reported to be only 1-2% of those produced by the same plasmid constructs in Bacillus subtilis.sup.4. The .beta.-galactosidase from Clostridium acetobutylicum.sup.5 was introduced into a lactococcal starter train, and enzyme activity detected. However, the maximum level of enzyme activity obtained was less than half of that measured in a wild type strain of L. lactis with innate .beta.-galactosidase activity. In all these instances no details are given of actual amounts of expressed protein present in the transformants. Expression of the bovine prochymosin.sup.6 gene in L. lactis has also been reported. Chymosin is an enzyme which is normally formed in the abomasum of young calves. It is a casein-specific protease used to curdle milk for cheese making. The gene encoding the precursor of chymosin (prochymosin) has been constitutively expressed in L. lactis using the promoter and secretion signal sequence of the proteinase gene of S. cremoris strain SK11; this work is the subject of European patent application number 88201203.2, filed on Jun. 13, 1988. The authors do not indicate the quantities of prochymosin produced in their expression strains of L. lactis but our inspection of the Western blots implies that the levels of expression obtained were low (estimated to be 0.2 mg/L supernatant). Only trace amounts of recombinant protein were detected in whole cell extracts.
Perhaps the best comparison with our own work is that which has been carried out on the pAC protein (a surface antigen) of Streptococcus mutans.sup.7. This was expressed in L. lactis by the introduction into L. lactis of a plasmid carrying the pAC gene within a 6.2 kb Sphl-BamHl DNA fragment derived from S. mutans. No attempt was made to control the expression of the gene. The yield of pAC protein in L. lactis was approximately 0.2% of dry weight, as compared to 1% in S. mutans. The pAC protein is secreted into culture supernatants of S. mutans to a level of approximately 5.5 mg/L. Because the pAC protein as produced in L. lactis lacked its cell membrane anchor domain it was anticipated that it would be efficiently secreted. However, this did not occur, the final yield of pAC protein in the L. lactis supernatants approached the limits of detection.